This application is the national phase under 35 U.S.C. xc2xa7371 of PCT International Application No. PCT/FI98/00238 which has an International filing date of Mar. 18, 1998 which designated the United States of America. This application also claims priority under 35 U.S.C. xc2xa7119(a)-(d) of prior foreign application Finland No. 971124 which has a filing date of Mar. 18, 1997.
The present invention relates to diagnosis of stomach cancer and concerns in specific a method for detection of gastric carcinoma at a premalignant phase by detecting cyclooxygenase-2 expression in a patient sample.
Gastric cancer is one of the most frequent and lethal malignancies in the world (Coleman et al., 1993). It is the fourth most common malignancy in Finnish males and the fifth in females, and accounts for 5% of all malignancies in Finland (Cancer Incidence in Finland 1994. Finnish Cancer Registry, Helsinki, 1996). Early detection of stomach cancer is difficult, and in most western countries the five year survival rate is less than 20% (Wanebo et al., 1993). More than 90% of stomach cancers are adenocarcinomas, which are divided into intestinal and diffuse types by the Lauren classification (Lauren, 1965).
Pathogenesis of gastric cancer is complex and incompletely understood, but in the case of intestinal type certain precursor changes, such as chronic atrophic gastritis, intestinal metaplasia, and epithelial dysplasia, have been associated with the disease (Antonioli, 1994). In contrast, the diffuse type lacks well-recognized precursor lesions. Since a different combination of genetic changes have been found in these two histologically distinct types of gastric cancer, they may possess different genetic backgrounds (Stemmermann et al., 1994; Tahara et al., 1996). However, malignancy related genetic changes shared by these two gastric adenocarcinoma types are thought to represent those that appear already at the premalignant phase of the disease (Tahara et al., 1996),
Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, indomethacin, and sulindac, inhibit chemically induced carcinoma of the colon in animal models (Steele et al., 1994; Giardiello et al., 1995). Epidemiological studies have shown that prolonged use of aspirin decreases incidence of and mortality from gastrointestinal cancers, including stomach cancer (Laakso et al., 1986; Giardiello, 1994; Thun, 1994; Thun et al., 1993).
The best known target of NSAIDs is cyclooxygenase (Cox), the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. Two Cox genes have been cloned (Cox-1 and Cox-2) that share over 60% identity at amino acid level and have similar enzymatic activities (Hershman, 1996; Smith et al., 1996). Cox-1 is considered as a housekeeping gene, and prostanoids synthesized via the Cox-1 pathway are thought to be responsible for cytoprotection of the stomach, for vasodilatation in the kidney, and for production of a proaggregatory prostanoid, thromboxane, by the platelets. In contrast, Cox-2 is an inducible immediate-early gene, and its pathophysiological role has been connected to inflammation, reproduction, and carcinogenesis.
Recent studies suggest that Cox-2 is connected to colon carcinogenesis and may thus be the target for the chemopreventive effect of NSAIDs: i) genetic disruption of Cox-2 gene or treatment with a Cox-2 specific drug suppress the polyp formation in a mice model for FAP (Oshima et al., 1996), ii) overexpression of Cox-2 in rat intestinal epithelial cells alters their rate of programmed cell death and their adhesion to the extracellular matrix (Tsujii et al., 1995), and iii) two different Cox-2-selective inhibitors suppress chemically induced aberrant crypt foci in the rat colon (Takahashi et al., 1996; Reddy et al., 1996). Further, elevated levels of Cox-2 mRNA and protein, but not those of Cox-1, are found in chemically induced rat colon carcinoma tissues (DuBois et al., 1996) and in human colon carcinoma, when compared to normal mucosa (Eberhart et al., 1994; Kargman et al., 1995; Sano et al., 1995).
The idea that chemopreventive effect of NSAIDs would be targeted against Cox-2, is further supported by the findings that Cox-2 selective compounds inhibit proliferation of rat intestinal epithelial cells, and mammary epithelial cells wherein Cox-2 expression was induced by oncogenes (Sheng et al., 1997 and Subbaramaiah et al. 1996). Also, Tsuji et al. (1996) reported recently that a Cox-2 specific inhibitor suppressed proliferation of a gastric and a colon carcinoma cell line, which expressed high steady-state levels of Cox-2 mRNA. This was not the case in cell lines that express low levels of Cox-2 mRNA.
Normal gastrointestinal tissues contain almost exclusively the Cox-1 isoform, and no functional Cox-2 protein was found in healthy stomach tissue (Kargman et al., 1996). Some Cox-2 mRNA may, however, be detected with more sensitive methods than the traditional Northern blot hybridization assay of total RNA, e.g. with RT-PCR (see O""Neill and Ford-Hutchinson 1993, and FIGS. 2 and 4 in the present work).
Since it is not known, whether Cox-2 is present in gastric cancer tissues in vivo, or in premalignant lesions of gastric carcinoma, we studied its expression in adenocarcinomas of the stomach, as well as in grave gastric dysplasias (which are highly premalignant). We found elevated levels of Cox-2 mRNA, but not those of Cox-1, in human gastric adenocarcinoma tissues and grave dysplasias of the stomach. However, the expression of Cox-2 was not elevated in mild dysplasias that rarely transform to malignancies. In gastric carcinoma, Cox-2 protein was primarily localized in the cancer cells.
Elevated expression of Cox-2 was not limited to the intestinal type, since each of the three diffuse carcinomas analysed contained higher levels of Cox-2 mRNA than their respective controls. Thus, overexpression of Cox-2 is one of the properties shared by these two histologically and genetically distinct diseases, which may suggest that it is involved with the early phase of carcinogenesis. Indeed, we found that Cox-2 is expressed in grave dysplasias of the stomach, whereas the expression was not elevated in mild dysplasias. This suggests that Cox-2 expression may be specifically associated with the premalignant lesions of gastric carcinoma.
In conclusion, we have shown that Cox-2 is expressed in human gastric carcinoma tissues, when compared to paired gastric mucosal specimens devoid of cancer cells. Cox-2 mRNA was found both in intestinal and diffuse adenocarcinomas. Cox-2 protein is localized to gastric carcinoma cells, but not to the surrounding stroma, as detected by immunohistochemistry. Importantly, grave gastric dysplasia specimens that represent premalignant lesions are strongly Cox-2 positive. This suggests that Cox-2 may be used as diagnostic marker of early gastric carcinoma, and in determination of the severity of premalignant lesions.
Expression of Cox-2 in human carcinomas seems, at least so far, to be restricted to the gastrointestinal tract. However, as colon carcinoma and stomach carcinoma are both epidemiologically, morphologically and genetically distinct diseases, the fact that elevated levels of Cox-2 mRNA and protein have been found in rodent and in human colon carcinoma tissues, does not give any indication of their role in gastric tissues. The fact that one gastric carcinoma cell line was shown to express high steady-state levels of Cox-2 mRNA, is neither any indication of its role in early gastric cancer in vivo.
The objective of this invention is to develop a method for diagnosis of early gastric carcinoma, which method comprises detection of Cox-2 mRNA or the Cox-2 protein in relevant patient samples. This is based on our finding that Cox-2 is highly expressed in gastric carcinoma cells and in premalignant lesions, but that its expression is very low or undetectable in normal stomach tissues.
The patient samples to be detected are e.g. biopsies or brush samples, which are obtained during routine gastroscopy or gastric lavage combined with brush technique. Gastric lavage and brush technique are well known methods in routine gastric cytology. These techniques provide cell samples from gastric mucosa for microscopic examination to include or exclude the possibility of malignancies in the stomach. Markers, such as Cox-2, may increase the sensitivity and specialty of the assay, when compared to the present method of just visualizing the morphology of the cells. Gastroscopic biopsy samples are either formalin fixed (for immunohistochemistry) or frozen in liquid nitrogen and stored in xe2x88x9270xc2x0 C. (for mRNA assays).
Cox-2 mRNA can be conveniently detected from said patient samples using methods known in the art. For instance, Northern blot analysis was shown by us to be extremely specific, and when combined to RT-PCR also very sensitive.
Cox-2 protein detection can be conveniently carried out from said patient samples using e.g. immunohistochemistry, which in addition to detection of Cox-2 expression shows the localization of the protein.
The present invention also provides test kits for carrying out the diagnostic method of the invention. Thus a kit for detection of Cox-2 mRNA expression comprises RNA or poly-A+mRNA isolation reagents, Cox-2 specific primers for RT-PCR and cDNA fragments for making either DNA or RNA (both sense and antisense) probes.
A kit for immunological detection of the Cox-2 protein comprises Cox-2 specific polyclonal or monoclonal antibodies. For peptide based analysis of Cox-2 protein a diagnostic kit is designed which comprises specific peptides with binding affinity to Cox-2. Such peptides are obtainable e.g. from phage display libraries. Also oligonucleotide based assays can be used, whereby oligonucleotides (modified RNA molecules) are included in a corresponding diagnostic kit.